1. Biochemical and structural exploration of the catalytic capacity of Sulfolobus KDG aldolases
- Author
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Wolterink-van Loo, Suzanne, van Eerde, Andre, Siemerink, Marco A. J., Akerboom, Jasper, Dijkstra, Bauke W., van der Oost, John, Akerboom, S.P., Agrotechnology and Food Sciences Group, Wageningen University and Research [Wageningen] (WUR), Groningen Biomolecular Sciences and Biotechnology, X-ray Crystallography, and Faculty of Science and Engineering
- Subjects
Models, Molecular ,substrate specificity ,Crystallography, X-Ray ,01 natural sciences ,Biochemistry ,chemistry.chemical_compound ,entner-doudoroff pathway ,Microbiologie ,Glyceraldehyde ,2-keto-3-deoxygluconate aldolase ,Cloning, Molecular ,0303 health sciences ,biology ,microbial ald ,Thermoplasma acidophilum ,Life Sciences ,dihydrodipicolinate synthase ,molecular replacement ,Sulfolobus ,ESCHERICHIA-COLI ,Crystallization ,Research Article ,Sulfolobus acidocaldarius ,thermophilic enzyme ,Sulfolobus tokodaii ,2-keto-3-deoxygluconate aldolase (KDGA) ,010402 general chemistry ,Microbiology ,MICROBIAL ALDOLASES ,03 medical and health sciences ,Escherichia coli ,Molecular replacement ,Binding site ,Pyruvates ,thermoplasma-acidophilum ,Molecular Biology ,Aldehyde-Lyases ,VLAG ,n-acetylneuraminate lyase ,030304 developmental biology ,Aldehydes ,Binding Sites ,Cell Biology ,carbon-carbon bond formation ,biology.organism_classification ,Lyase ,hyperthermophilic archaea ,0104 chemical sciences ,chemistry ,2-keto-3-deoxy-6-phosphogluconate kdpg aldolase - Abstract
Aldolases are enzymes with potential applications in biosynthesis, depending on their activity, specificity and stability. In the present study, the genomes of Sulfolobus species were screened for aldolases. Two new KDGA [2-keto-3-deoxygluconate (2-oxo-3-deoxygluconate) aldolases] from Sulfolobus acidocaldarius and Sulfolobus tokodaii were identified, overexpressed in Escherichia coli and characterized. Both enzymes were found to have biochemical properties similar to the previously characterized S. solfataricus KDGA, including the condensation of pyruvate and either D,L-glyceraldehyde or D,L-glyceraldehyde 3-phosphate. The crystal structure of S. acidocaldarius KDGA revealed the presence of a novel phosphate-binding motif that allows the formation of multiple hydrogen-bonding interactions with the acceptor substrate, and enables high activity with glyceraldehyde 3-phosphate. Activity analyses with unnatural substrates revealed that these three KDGAs readily accept aldehydes with two to four carbon atoms, and that even aldoses with five carbon atoms are accepted to some extent. Water-mediated interactions permit binding of substrates in multiple conformations in the spacious hydrophilic binding site, and correlate with the observed broad substrate specificity.
- Published
- 2006